Pseudomnonas syringae pv. tomato DC3000 is a widely studied model plant pathogen that causes disease on tomato and Arabidopsis. DC3000 uses a type III secretion (TTSS) system to directly deliver bacterial effector proteins into the host cell (Galan et al., “Type III secretion machines: Bacterial devices for protein delivery into host cells.” Science, 284: 1322-1328 (1999)). Loss of function mutations in the TTSS completely abrogate P. syringae disease formation, indicating that effectors are essential agents of P. syringae pathogenesis (Collmer et al., “Pseudomonas syringae Hrp type III secretion system and effector proteins.” Proc Natl Acad Sci USA, 97: 8770-8777 (2000)). In bacterial pathogens of plants, the TISS is encoded by the hypersensitive response (“HR”) and pathogenicity (hrp) genes (Lindgren, P. B., “The role of hrp genes during plant-bacterial interactions.” Annu. Rev. Phytopathol. 35: 129-152 (1997)). Mutations in key hip genes prevent the secretion of effectors and inhibit pathogen growth and host defenses. A hallmark of effector genes is the presence of a “Hrp box” cis element in their promoter which is recognized by the HrpL ECF-like sigma factor (Innes et al., “Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes. ” J. Bacteriol. 175: 4859-4869 (1993); Xiao et al., “Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonase syringae pv. syringae Pss61 hrp and hrmA genes.” J. Bacteriol. 176: 1025-1036 (1994)). A recent search for Hrp box containing genes in the genome of Pseudomonas syringae pv. tomato strain DC3000 revealed over 20 putative effector genes (Fouts, et al., “Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.” Proc Natl Acad Sci USA, 99: 2275-2280 (2002)). Although the role of effector proteins in pathogen virulence is poorly understood, many effectors have been isolated based on their ability to trigger host immunity.
In the “gene-for-gene” model of plant immunity, disease resistance is initiated by recognition of a pathogen avirulence (Avr) effector protein by a plant resistance (R) protein. The tomato R protein Pto, a serine/threonine protein kinase, recognizes and directly interacts with DC3000 effector proteins AvrPto and AvrPtoB, and initiates immunity in tomato by characterized and uncharacterized signaling mechanisms (Kim et al., “Two distinct pseudomonas effector proteins interact with the pto kinase and activate plant immunity.” Cell, 109: 589-598 (2002); Scofield et al., “Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato.” Science, 274: 2063-2065 (1996); Sessa et al., “Signal recognition and transduction mediated by the tomato Pto kinase: a paradigm of innate immunity in plants.” Microbes Infect, 2: 1591-1597 (2000); Tang et al., “Overexpression of Pto activates defense responses and confers broad resistance.” Plant Cell, 11: 15-30 (1999)). Interestingly, the Pto kinase shares sequence similarity with the human interleukin-1 receptor associated kinase (IRAK) and with the Drosophlila Pelle kinase, both of which, like Pto, play a role in immune responses (Cohn et al., “Innate immunity in plants.” Curr. Opin. Immunol., 13: 55-62 (2001); Hoffman et al., “Phylogenetic perspectives in innate immunity,” Science 284:1313-1318 (1999)). The Pto gene belongs to a gene family of 6 members on tomato chromosome 5 (Martin et al., “Map-based cloning of a protein kinase gene conferring disease resistance in tomato.” Science, 262: 1432-1436 (1993); Michelmore et al., “Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process.” Genome Res. 8: 1113-1130 (1998); Riely et al., “Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto.” Proc. Natl. Acad. Sci. USA 98: 2059-2064 (2001)). One of these family members, Fen, encodes a kinase that confers sensitivity to an insecticide (fenthion), while the function of the others is unknown (Martin et al., “A Member of Tomato Pto Gene Family Confers Sensitivity to Fenthion Resulting in Tomato,” Plant Cell 6:1543-1552 (1994)).
The R gene-mediated plant immune response is characterized by a series of physiological changes in the plant cell, including the formation of reactive oxygen species, induction of defense genes, and the HR. The HR is defined as a defense response involving rapid, localized cell death that functions to limit pathogen growth (Goodman et al., “The hypersensitive reaction in plants to pathogens.” APS Press, St. Paul, Minn., USA, (1994)). The cell death associated with the HR is a genetically controlled and regulated process and an example of programmed cell death in plants (Greenberg, J. T. “Programmed cell death in plant-pathogen interactions.” Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 525-545 (1997); Heath, M. C. “Hypersensitive response-related death.” Plant Mol Biol, 44: 321-334 (2000)). As such, programmed cell death is a hallmark of HR-based immunity in plants, and cell death phenotypes are often used in laboratory experiments to discover and dissect plant immune responses.
The AvrPtoB protein has a predicted molecular mass of 59 kDa, is secreted via the TTSS, and triggers the HR and immunity in Pto-expressing tomato plants. AvrPtoB has limited similarity to AvrPto; however, it shares 52% amino acid identity with the P. s. pv. phaseolicola effector VirPphA (Jackson et al., “Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola.” Proc Natl Acad Sci USA, 96: 10875-10880 (1999)). In general, bacterial effector proteins are highly diverse with little amino acid sequence similarity among them (one exception is the AvrBs3 family; Lindgren, P. B., “The role of hrp genes during plant-bacterial interactions.” Annu. Rev. Phytopathol. 35: 129-152 (1997); White et al., “Prospects for understanding avirulence gene function.” Curr. Opin. Plant Biol. 3: 291-298 (2000)). They have been identified from all four of the most common genera of plant bacterial pathogens (i.e., Pseudomonas, Xanthomonzas, Erwinia, and Ralstonia). In a still cryptic process, these pathogens utilize the TTSS to inject effectors across the plant cell wall into the cytoplasm (Galan et al., “Type III secretion machines: Bacterial devices for protein delivery into host cells.” Science, 284: 1322-1328 (1999); Jin et al., “Role of the Hrp pilus in type III protein secretion in Pseudomonas syringae.” Science 294: 2556-2558 (2001)). Little is known of the fate of bacterial effectors once they are in the plant cell although some members of the AvrBs3 family are localized to the nucleus, some effector proteins are targeted to the plasma membrane after being myristylated, and others are processed to smaller forms (Nimchuk et al., “Eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type III effector proteins from Pseudomonas syringae.” Cell. 101: 353-363 (2000); Shan et al., “The Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane.” Plant Cell 12: 2323-2337 (2000b); Van der Ackerveken et al., “Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host cell.” Cell 87: 1307-1316 (1996); Zhu et al., “The C terminus of AvrXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus.” Plant Cell. 11: 1665-1674 (1999)).
The AvrPto protein and the Pto kinase physically interact in a yeast two-hybrid system (Scofield et al., “Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato.” Science 274: 2063-2065 (1996); Tang et al., “The avirulence protein AvrPto physically interacts with the Pto kinase.” Science 274: 2060-2063 (1996)). Co-expression of Pto and AvrPto as transgenes in a pto mutant leaf is sufficient to activate resistance. Mutations that disrupt this interaction also abolish the ability to elicit disease resistance in plant leaves (Chang et al., “Functional studies of the bacterial avirulence protein AvrPto by mutational analysis.” Mol. Plant-Microbe Interact. 14: 451-459 (2001); Frederick et al., “Recognition specificity for the bacterial avirulence protein AvrPto is determined by Thr-204 in the activation loop of the tomato Pto kinase.” Molecular Cell. 2: 241-245 (1998); Shan et al., “The Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane.” Plant Cell 12: 2323-2337 (2000)). Resistance is dependent on the Prf protein which bears striking similarity to the large NB-LRR class of R proteins (Salneron et al., “Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster.” Cell 86: 123-133 (1996)). Pto-Fen chimeras were used to define the kinase activation loop as a key determinant of Pto interaction specificity for AvrPto (Frederick et al., “Recognition specificity for the bacterial avitulence protein AvrPto is determined by Thr-204 in the activation loop of the tomato Pto kinase.” Molecular Cell. 2: 241-245 (1998); Scofield et al., “Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato.” Science 274: 2063-2065 (1996); Tang et al., “The avirulence protein AvrPto physically interacts with the Pto kinase.” Science 214: 2060-2063 (1996)). Pto kinase is phosphorylated on 8 residues and mutation of two of these residues (T38 and S198) abolishes its ability to elicit host resistance (Sessa et al., “Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto-Pto-mediated hypersensitive response.” EMBO J. 19: 2257-2269 (2000)). Recognition specificity of Pto for AvrPto appears to have evolved before Lycopersicon speciation because a Pto family member from a distantly related species, L. hirsutum, also recognizes AvrPto (Riely et al., “Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto.” Proc. Natl. Acad. Sci. USA 98: 2059-2064 (2001)).
The AvrPto gene was originally isolated from P. s. tomato strain JL1065 based on its ability to confer avirulence to a virulent strain of P. s. inaculicola (Ronald et al., “The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene.” 174: 1604-1611 (1992)). AvrPto encodes an 18 kD protein that bears little sequence similarity to proteins in current databases (Salmeron et al., “Molecular characterization and hrp dependence of the avirulence gene avrPto from Pseudomoizas syringae pv. tomato.” Mol. Gen. Genet. 239: 6-16 (1993)). Its mechanism of activating resistance is unknown although it likely interacts with Pto inside the plant cell and possibly with certain ‘AvrPto-dependent Pto-interacting’ (Adi) proteins as part of a complex (Bogdanove et al., “AvrPto-dependent Pto-interacting proteins and AvrPto-interacting proteins in tomato.” Proc. Natl. Acad. Sci. USA 97: 8836-8840 (2000); Scofield et al., “Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato.” Science 274: 2063-2065 (1996); Tang et al., “The avirulence protein AvrPto physically interacts with the Pto kinase.” Science 274: 2060-2063 (1996)). AvrPto acts as a virulence factor when Pto (or Prf) is absent from the plant cell and increases the growth of P. s. tomato about 10-fold as compared to a strain lacking the effector (Chang et al., “avrPto enhances growth and necrosis caused by Pseudomonas syringae pv. tomato in tomato lines lacking either Pto and Prf.” Mol. Plant-Microbe Interact. 13: 568-571 (2000); Shan et al., “A cluster of mutations disrupt the avirulence but not the virulence function of AvrPto.” Mol. Plant-Microbe Interact. 13: 592-598 (2000)). In common with several effectors, AvrPto has a myristylation motif at its N terminus that is required for both its avirulence and virulence activity (Nimchuk et al., “Eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type III effector proteins from Pseudomonas syringae.” Cell. 101: 353-363 (2000); Shan et al., “The Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane.” Plant Cell 12: 2323-2337 (2000)). The amino acids of AvrPto that are required for its recognition by the Pto kinase have been examined by saturation mutagenesis (Chang et al., “Functional studies of the bacterial avirulence protein AvrPto by mutational analysis.” Mol. Plant-Microbe Interact. 14: 451-459 (2001); Shan et al., “A cluster of mutations disrupt the avirulence but not the virulence function of AvrPto.” Mol. Plant-Microbe Interact. 13: 592-598 (2000); Shan et al., “The Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane.” Plant Cell 12: 2323-2337 (2000)). Mutation of three AvrPto residues—S94, 196, and G99—abolishes interaction with Pto and avirulence activity, but not virulence activity, in tomato (Shan et al., “A cluster of mutations disrupt the avirulence but not the virulence function of AvrPto.” Mol. Plant-Microbe Interact. 13: 592-598 (2000); Shan et al., “The Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane.” Plant Cell 12: 2323-2337 (2000)). Along with the other observations (Chang et al., “Functional studies of the bacterial avirulence protein AvrPto by mutational analysis.” Mol. Plant-Microbe Interact. 14: 451-459 (2001)), these results indicate that an internal region of AvrPto determines its binding specificity for Pto.
AvrPto-like DNA sequences are present in Pseudomonas strains that are known to be avirulent on Pto tomato plants (race 0 strains) and are absent from virulent ones (race 1 strains; Ronald et al., “The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene.” 174: 1604-1611 (1992)). Thus, a homolog of avrPto was identified in avirulent P. s. tomato strain DC3000 based on DNA blot hybridization (Ronald et al., “The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene.” 174: 1604-1611 (1992)). Gene replacement strains in which the avrPto reading frame was deleted were constructed in strains JL1065 and DC3000. Surprisingly, both mutant strains were still recognized by Pto-expressing tomato leaves (Ronald et al., “The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene.” 174: 1604-1611 (1992)). A later study found that a tomato line carrying a CaMV 35S::Pto transgene (and not a sibling line without Pto) is resistant to the avrPtoΔDC3000 deletion strain. These results implied that strains DC3000 and JL1065 carry additional avirulence proteins that are recognized specifically by Pto.
In recent years, evidence has accumulated that effector proteins can interfere with host defense responses. In a breakthrough study, Jackson et al., “Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola.” Proc Natl Acad Sci USA, 96: 10875-10880 (1999) demonstrated that VirPphA allows P. s. pv. phaseolicola to evade HR-based immunity in bean. Other P. s. pv. phaseolicola effectors also allow the pathogen to avoid triggering host immunity, including AvrPphC and AvrPphF (Tsiamis et al., “Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease.” Embo J, 19: 3204-3214 (2000)). Additionally, in the P. s. pv. maculicola-Arabidopsis pathosystem, interference has been observed with the effector proteins AvrRpt2 and AvrRpm1 and the HR initiated by the R proteins RPS2 and RPM1, respectively (Reuber et al, “Isolation of arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes.” Plant Cell, 8: 241-249 (1996); Ritter et al., “Interference between two specific pathogen recognition events mediated by distinct plant disease resistance genes.” Plant Cell, 8: 251-257 (1996)). These findings suggest that for some effector proteins virulence activity can be dominant over avirulence activity. Although the phenomenon of effector-mediated evasion of plant immunity has been well documented, the molecular basis of this activity has remained mysterious. Several hypotheses have been proposed to explain how some effector proteins (such as VirPphA, AvrPphC and AvrPphF) prevent a host from detecting a pathogen, including: i) inhibition of avr gene expression; ii) blocking of Avr protein secretion or translocation; iii) interference with Avr/R protein recognition inside the plant cell; or iv) suppression of HR or disease resistance signaling downstream of Avr recognition (Jackson et al., “Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola.” Proc Natl Acad Sci USA, 96: 10875-10880 (1999); Tsiamis et al., “Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease.” Embo J, 19: 3204-3214 (2000)). Specific support, however, for any one of these hypotheses has not been reported.
The present invention is directed to overcoming these and other deficiencies in the art.